Quantitation of the PD-1/PD-L1 axis in non-small cell lung cancer by immuno-multiple reaction monitoring.
Authors: Vincent Lacasse, Rene P. Zahedi, Vincent R. Richard, Hangjun Wang, Georgia Mitsa, Olivier Poetz, Margaret Redpath, Andreas I. Papadakis, Mounib Elchebly, Victor Cohen, Jason S. Agulnik, Gerald Batist, Christoph H. Borchers, Alan Spatz
Journal: Journal of Clinical Oncology (2023)

e14656 Background: Immuno-oncology revolutionized lung cancer treatment, but predictive biomarkers of response to checkpoint inhibitors (CPI) are still lacking. Only 50% of PD-L1 positive tumors by immunohistochemistry (IHC) respond to anti-PD-L1 treatment. Analytical variability and post-translational modifications (PTM) of the PD-1 signaling associated proteins (e.g.: glycosylation), can explain some of this discrepancy. Plus, the tumor immune microenvironment (TME) is complex and PD-L1 IHC alone is a flawed surrogate its status. Mass spectrometry based technologies have the potential to overcome these challenges by integrating sensitivity, specificity and absolute quantitation of proteins and PTMs in a standardized fashion. Here, we demonstrate the advantages of using anti-peptide antibodies to purify surrogate peptides followed by liquid-chromatography (LC) and multiple reaction monitoring (MRM), herby termed as iMRM, to gain new insight into the TME. Methods: To determine the concentration of PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70, we used unique and well detectable proteolytic peptides as surrogates. Our previously described protocol (ASCO 2022, #377181) allows robust quantitation of 13 peptides and monitors the glycosylation status of PD-L1, PD-L2, and PD-1. NSCLC samples were either fresh frozen (n = 42) or FFPE (n = 77) and sometimes both (n = 17). PD-L1 quantitation by iMRM was compared to PD-L1 IHC 22C3. Results: This multiplexed iMRM assay successfully quantified the PD-1/PD-L1 axis proteins in 96% of NSCLC patients (61/63). PD-L1 glycosylation levels ranged from 82 to 100% (n = 69, median = 100%, SD = 3.7%). Only PD-1 was significantly different between the fresh frozen (mean = 11±6 amol/µg total protein) and FFPE group (mean = 5±2 amol/µg total protein, ρ = 0.0001), all other peptides showed comparable levels. In our 17 matched FF/FFPE samples, PD-L1 moderately correlated (R = 0.45, ρ = 0.045) and the other peptides did not correlate (R &lt; 0.03). Intra/intertumoral heterogeneity, differences in cellularity and tumor origin likely contributed to this discrepancy. IMRM results correlated moderately (R = 0.56, ρ &lt; 0.01) with PD-L1 IHC. Most of these patients were not CPI-treated, but survival data was available. A trend was noted between the concentration of certain targets and patient survival. Linear regression was used to establish a cut-off for each peptide from which an immunoscore was calculated. The immunoscore could predict long-term survival (accuracy 75.4%) irrespective of tumor staging, grade or subtype and survival was significantly associated with the immunoscore (log-rank ρ = 0.005). Conclusions: Our iMRM workflow provides a new understanding of the TME in NSCLC through the PD-1/PD-L1 axis with an easy-to-read immunoscore. A set of 60 tumors from CPI-treated patients is currently being processed to validate the clinical utility of the assay in relation with CPI-response.